In general a photonic biosensor array, sometimes called microarray or biochip, comprises a collection of probe spots to which different targets may attach. For example in the case of a DNA microarray the probes are oligonucleotides, cDNA or similar which are hybridised with fluorescence labelled samples, typically of two colours, one for the patient the other for the control. Fluorescence from the hybridised array is then viewed to determine to which spots binding has occurred. There are other types of array such as protein arrays (including antibody arrays) where spots of protein molecules (or antibodies) are used to identify the complementary entity (antibodies or proteins). Thus chemical compound arrays may be employed to search for proteins and other biologically active molecules again by employing functionalising molecules or entities in an array of spots which bind with specific biological targets. In general, however, all these techniques employ fluorescence labelling of the targets to detect binding events on the array.
By contrast the techniques we describe here do not employ fluorescence but instead rely upon plasmon resonance-based sensing. Broadly speaking in this technique total internal reflection of light is used to generate an evanescent wave which excites plasmons (a collective electronic excitation) in a metallic conductor, which are modified by the presence of a target molecule on the surface of the conductor. The modification results in a shift, generally in both wavelength and amplitude, of the plasmon resonance peak detectable in the totally internally reflected light. Plasmon resonance-based sensing has the ability to detect very small changes in the effective refractive index in a medium adjacent the surface of the metallic conductor, for example down to Δn of the order of 10−4 refractive index units (RIU).
It is known to employ label-free surface plasmon resonance (SPR) based technology for studying biomolecular interaction in real time and, in particular, technology for this is available from the Swedish company BIAcore AB; for background technical information see published BIAcore patent applications such as WO 2006/135309, WO 94/00751, U.S. Pat. No. 4,997,278, and WO 97/19375. However BIAcore employ a continuous metal surface. Some further technical background information relating to plasmon resonance-based sensing in a different context (evanescent wave cavity ringdown spectroscopy) can be found in Evanes Co patent application WO 2005/088277.
There is, however, a need to provide improved microarray assay techniques, in particular with increased sensitivity. The applicants have recognised that, in principle, refractive index changes of orders of magnitude better than 10−4 RIU are potentially possible. Such increased sensitivity could provide a doctor with a great deal of information for help in diagnosing a pathological condition, especially if techniques could be found to address the non-specific binding events which could swamp any genuine signal at high sensitivities. Embodiments of the techniques we describe provide a step towards solutions of these problems.